TOXICITY OF SEVERAL FUNGICIDES FOR ORANGE WHEAT BLOSSOM MIDGE, SITODIPLOSIS MOSELLANA (GÉHIN) (DIPTERA: CECIDOMYIIDAE).

The orange wheat blossom midge, Sitadiplosis mosellano (Géhin) (Diptera: Cecidomyiidae), can cause severe losses in wheat grain yield and quality. This pest is known to be susceptible to many insecticides, but various field observations have suggested that some fungicides could also significantly affect S. mosellana. In order to confirm these field observations, the effect on adult midges of several fungicides commonly applied to wheat crops was investigated in the laboratory and in small plots in the field. In each experiment, the fungicides were compared with a positive (insecticide) and a negative control (water). Four fungicides were assessed in the laboratory, each with five doses based on basis of a tenfold dilution starting at the field-recommended dose. The mortality rate was evaluated after 24 hours and the lethal dose 50% (LD50) was determined for each product. In the field, six fungicides were tested at the recommended dose. The effect of each product was compared on the basis of the number of S. mosellana adults caught alive with an insect vacuum sampler (Vortis®) on the morning after the treatments. Both experiments showed a significant effect of several fungicides tested on S. mosellana adults. Chlorothalonil was not toxic for S. mosellana, but tebuconazole, fluxapyroxad and azoxystrobin all induced significant mortality rates.

Unusual occurrence of cocoons in population of Haplodiplosis marginata (Diptera: Cecidomyiidae) in Belgium.

The saddle gall midge, Haplodiplosis marginata (von Roser) (Diptera: Cecidomyiidae), is a phytophagous species that develops in saddle-shaped galls on stems of wheat Triticum vulgare, barley Hordeum sativum, rye Secale cereale, and some other species of Poaceae. Only one generation develops per year. Full-grown larvae leave galls and drop onto the soil where they remain up to the springtime of the following year. Larvae do not usually spin cocoons. However, formation of cocoons by larvae was observed in populations developing in western Europe: in England in 1954, in the Netherlands in the 1960s, and in Belgium in 2011. On the basis of our analysis, a part of the larval population forms cocoons as protection against unfavorable weather conditions, especially drought.

Study on the sensitivity of three oat varieties to the saddle gall midge, haplodiplosis margina ta (von Roser) (Diptera: cecidomyiidae).

The saddle gall midge, Haplodiplosis marginota (von Roser, 1840) is a univoltine pest of cereals which occurs in Europe. The larvae feed on stems and attractive saddle-shaped depressions, driving to important yield losses when the galls are numerous. After 40 years without any reporting, large populations of H. marginata and important damage have been observed since 2010 in wheat crops in Belgium, especially in the Flemish Polders where clay soils and intensive farming of cereals favour heavy infestations. According to some research conducted in the 1960s during the last outbreak, oat (Avena sativa L.) is known to be one of the less favourable hosts to the saddle gall midge. Our study was performed in order to assess the host sensitivity of three oat varieties currently grown in Belgium: EVITA, EFFEKTIv and FREDDY. Therefore, oat varieties were sown on infested soil in two separate enclosures in a glasshouse. In the first enclosure, only the three oat varieties were grown; in the second one, these three oat varieties were grown together with two varieties of spring wheat: GRANNY and KWS CHAMSIN. TWO parameters were measured: the percentage of leaves with laid eggs, and the number of galls per stem. The percentage of leaves with eggs showed that the infestation was significantly lower on oats when they were in presence of wheat. The egg infestation was also significantly higher on wheat than on oat, which means oat is a much less favourable host plant than spring wheat for egg laying. Oat varieties were significantly different from each other regarding the number of galls per stem, but with very little damage compared to wheat. The FREDDY variety even seemed to be completely resistant to saddle gall midge, as no galls were observed although there was a similar percentage of leaves with eggs for the three oat varieties. Cropping oat could thus contribute to reduce infestations of H. morginato.

Integrated management of wild chamomile (Matricaria chamomilla L.) populations by tillage.

Nowadays, environmental, health and economic concerns encourage reviewing our weed management in agriculture. Integrated pest management is one key element in the development of weed management strategies less dependent from herbicides. To reach this goal, impact of different methods of tillage (Combinations of stubble cultivator and moldboard plow) on biology and dynamic of wild chamomile populations was studied in experimental plots of experimental farm of Gembloux Agro-Bio Tech. In summer 2012, wild chamomile densities were significantly lower in plots tilled with a moldboard plow. The use of a stubble cultivator did not significantly affect M. chamomilla density. In addition, we found higher wheat yields in plowed plots, indicating that the decrease in M. chamomilla densities reduces competition for wheat. These results show well long run impact of plowing and his effect on densities of wild chamomile and the seedbank.

Chemical control of Haplodiplosis marginata von Roser (Diptera: Cecidomyiidae).

The saddle gall midge, Haplodiplosis marginata (von Roser), has been detected in Belgium since 2010, after several decades without any reporting. It had indeed caused serious damages between 1965 and 1970. This insect is a European cereal pest whose larvae feed on stems and engender saddle-shaped depressions, resulting in yield losses. Face with the resurgence of this pest, it was decided to study its spatial distribution and, because serious damages were observed in some regions, to develop effective curative control. To date, chemical protection seems to be the only immediate solution in case of heavy emergences. Experimentation was conducted in a highly infested field (Meetkerke, Belgian Polders), according to a randomized complete blocks arrangement with four replications. Foremost, a lambdacyhalothrin-based insecticide was used to evaluate efficiency of several protection schemes, ranging between one and four spray(s). The large spread of flights observed during the 2011 spring allowed to highlight the effect of treatment date on the attack intensity and also on the galls distribution along the stem, on the different internodes: the lower internodes were protected by the early sprayings, while last sprayings induced reduction of galls number on the upper internodes. Moreover, several insecticides already registrated in cereals against aphids were compared for their efficacy against saddle gall midge. Studied pyrethroids have shown a very good efficacy, ranging between 75% and 87%, when applied twice with a 2 weeks interval. To be efficient, insecticide applications must thus be synchronized with the flights and egg-laying periods. Monitoring the phenology of flights is thus essential as part of integrated pest management against saddle gall midge.

Effect of mechanical weeding on wild chamomile (Matricaria chamomilla L.) populations in winter wheat crop (Triticum aestivum L.).

Currently, economic, agronomic and environmental concerns lead to reduce the use of herbicides. Mechanical weeding can help to reach this objective. Dynamics and biology of wild chamomile (Matricaria chamomilla L.) populations were assessed as well as dynamic of winter wheat (Triticum aestivum L.) for four level of application of a weeder-harrow (0, 1, 2, 3 treatment(s)). After each treatment, an effect of mechanical weeding on wild chamomile density was observed. Density of wild chamomile decreased significantly with intensification of mechanical weeding. A third treatment allowed eliminating late emerged plants.

Dynamic of black-grass populations depending on the sowing time of winter wheat.

Currently, economic, agronomic and environmental concerns, lead to reduce use of herbicides. This reduction can be help by cultural measures like delay of the sowing date. Four sowing dates of winter wheat from 15th of October to 26th of November were tested. Dynamic of black-grass (Alopecurus myosuroides Huds.) populations and their reproduction rate were assessed as well as dynamic of winter wheat for each date. Delay of sowing could significantly reduce reproduction rate of black-grass. It was shown that the emergence rate (pl/m2), but also number of ears per plant and number of seeds per ear of black-grass decreased significantly with the sowing date. This reduction of seeds production already is from sixty per cent for a delay of two weeks sowing.

Diel shifts in carboxylation pathway and metabolite dynamics in the CAM bromeliad Aechmea 'Maya' in response to elevated CO2.

BACKGROUND AND AIMS: The deployment of temporally separated carboxylation pathways for net CO(2) uptake in CAM plants provides plasticity and thus uncertainty on how species with this photosynthetic pathway will respond to life in a higher-CO(2) world. The present study examined how long-term exposure to elevated CO(2) influences the relative contributions that C(3) and C(4) carboxylation make to net carbon gain and to establish how this impacts on the availability of carbohydrates for export and growth and on water use efficiency over the day/night cycle. METHODS: Integrated measurements of leaf gas exchange and diel metabolite dynamics (e.g. malate, soluble sugars, starch) were made in leaves of the CAM bromeliad Aechmea 'Maya' after exposure to 700 micromol mol(-1) CO(2) for 5 months. KEY RESULTS: There was a 60 % increase in 24-h carbon gain under elevated CO(2) due to a stimulation of daytime C(3) and C(4) carboxylation in phases II and IV where water use efficiency was comparable with that measured at night. The extra CO(2) taken up under elevated CO(2) was largely accumulated as hexose sugars during phase IV and net daytime export of carbohydrate was abolished. Under elevated CO(2) there was no stimulation of dark carboxylation and nocturnal export and respiration appeared to be the stronger sinks for carbohydrate. CONCLUSIONS: Despite the increased size of the soluble sugar storage pool under elevated CO(2), there was no change in the net allocation of carbohydrates between provision of substrates for CAM and export/respiration in A. 'Maya'. The data imply the existence of discrete pools of carbohydrate that provide substrate for CAM or sugars for export/respiration. The 2-fold increase in water-use efficiency could be a major physiological advantage to growth under elevated CO(2) in this CAM bromeliad.

Repressors and upstream repressing sequences of the stress-regulated ENA1 gene in Saccharomyces cerevisiae: bZIP protein Sko1p confers HOG-dependent osmotic regulation.

The yeast ENA1/PMR2A gene encodes a cation extrusion ATPase in Saccharomyces cerevisiae which is essential for survival under salt stress conditions. One important mechanism of ENA1 transcriptional regulation is based on repression under normal growth conditions, which is relieved by either osmotic induction or glucose starvation. Analysis of the ENA1 promoter revealed a Mig1p-binding motif (-533 to -544) which was characterized as an upstream repressing sequence (URSMIG-ENA1) regulated by carbon source. Its function was abolished in a mig1 mig2 double-deletion strain as well as in either ssn6 or tup1 single mutants. A second URS at -502 to -513 is responsible for transcriptional repression regulated by osmotic stress and is similar to mammalian cyclic AMP response elements (CREs) that are recognized by CREB proteins. This URSCRE-ENA1 element requires for its repression function the yeast CREB homolog Sko1p (Acr1p) as well as the integrity of the Ssn6p-Tup1p corepressor complex. When targeted to the GAL1 promoter by fusing with the Gal4p DNA-binding domain, Sko1p acts as an Ssn6/Tup1p-dependent repressor regulated by osmotic stress. A glutathione S-transferase-Sko1 fusion protein binds specifically to the URSCRE-ENA1 element. Furthermore, a hog1 mitogen-activated protein kinase deletion strain could not counteract repression on URSCRE-ENA1 during osmotic shock. The loss of SKO1 completely restored ENA1 expression in a hog1 mutant and partially suppressed the osmotic stress sensitivity, qualifying Sko1p as a downstream effector of the HOG pathway. Our results indicate that different signalling pathways (HOG osmotic pathway and glucose repression pathway) use distinct promoter elements of ENA1 (URSCRE-ENA1 and URSMIG-ENA1) via specific transcriptional repressors (Sko1p and Mig1/2p) and via the general Ssn6p-Tup1p complex. The physiological importance of the relief from repression during salt stress was also demonstrated by the increased tolerance of sko1 or ssn6 mutants to Na+ or Li+ stress.

The Sko1p repressor and Gcn4p activator antagonistically modulate stress-regulated transcription in Saccharomyces cerevisiae.

In the transcriptional response of Saccharomyces cerevisiae to stress, both activators and repressors are implicated. Here we demonstrate that the ion homeostasis determinant, HAL1, is regulated by two antagonistically operating bZIP transcription factors, the Sko1p repressor and the Gcn4p activator. A single CRE-like sequence (CRE(HAL1)) at position -222 to -215 with the palindromic core sequence TTACGTAA is essential for stress-induced expression of HAL1. Down-regulation of HAL1 under normal growth conditions requires specific binding of Sko1p to CRE(HAL1) and the corepressor gene SSN6. Release from this repression depends on the function of the high-osmolarity glycerol pathway. The Gcn4p transcriptional activator binds in vitro to the same CRE(HAL1) and is necessary for up-regulated HAL1 expression in vivo, indicating a dual control mechanism by a repressor-activator pair occupying the same promoter target sequence. gcn4 mutants display a strong sensitivity to elevated K(+) or Na(+) concentrations in the growth medium. In addition to reduced HAL1 expression, this sensitivity is explained by the fact that amino acid uptake is drastically impaired by high Na(+) and K(+) concentrations in wild-type yeast cells. The reduced amino acid biosynthesis of gcn4 mutants would result in amino acid deprivation. Together with the induction of HAL1 by amino acid starvation, these results suggest that salt stress and amino acid availability are physiologically interconnected.

CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae.

The expression of gluconeogenic fructose-1,6-bisphosphatase (encoded by the FBP1 gene) depends on the carbon source. Analysis of the FBP1 promoter revealed two upstream activating elements, UAS1FBP1 and UAS2FBP1, which confer carbon source-dependent regulation on a heterologous reporter gene. On glucose media neither element was activated, whereas after transfer to ethanol a 100-fold derepression was observed. This gene activation depended on the previously identified derepression genes CAT1 (SNF1) (encoding a protein kinase) and CAT3 (SNF4) (probably encoding a subunit of Cat1p [Snf1p]). Screening for mutations specifically involved in UAS1FBP1 derepression revealed the new recessive derepression mutation cat8. The cat8 mutants also failed to derepress UAS2FBP1, and these mutants were unable to grow on nonfermentable carbon sources. The CAT8 gene encodes a zinc cluster protein related to Saccharomyces cerevisiae Gal4p. Deletion of CAT8 caused a defect in glucose derepression which affected all key gluconeogenic enzymes. Derepression of glucose-repressible invertase and maltase was still normally regulated. A CAT8-lacZ promoter fusion revealed that the CAT8 gene itself is repressed by Cat4p (Mig1p). These results suggest that gluconeogenic genes are derepressed upon binding of Cat8p, whose synthesis depends on the release of Cat4p (Mig1p) from the CAT8 promoter. However, gluconeogenic promoters are still glucose repressed in cat4 mutants, which indicates that in addition to its transcription, the Cat8p protein needs further activation. The observation that multicopy expression of CAT8 reverses the inability of cat1 and cat3 mutants to grow on ethanol indicates that Cat8p might be the substrate of the Cat1p/Cat3p protein kinase.

Glucose derepression of gluconeogenic enzymes in Saccharomyces cerevisiae correlates with phosphorylation of the gene activator Cat8p.

The Cat8p zinc cluster protein is essential for growth of Saccharomyces cerevisiae with nonfermentable carbon sources. Expression of the CAT8 gene is subject to glucose repression mainly caused by Mig1p. Unexpectedly, the deletion of the Mig1p-binding motif within the CAT8 promoter did not increase CAT8 transcription; moreover, it resulted in a loss of CAT8 promoter activation. Insertion experiments with a promoter test plasmid confirmed that this regulatory 20-bp element influences glucose repression and derepression as well. This finding suggests an upstream activating function of this promoter region, which is Mig1p independent, as delta mig1 mutants are still able to derepress the CAT8 promoter. No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression. Fusions of Cat8p with the Gal4p DNA-binding domain mediated transcriptional activation. This activation capacity was still carbon source regulated and depended on the Cat1p (Snf1p) protein kinase, which indicated that Cat8p needs posttranslational modification to reveal its gene-activating function. Indeed, Western blot analysis on sodium dodecyl sulfate-gels revealed a single band (Cat8pI) with crude extracts from glucose-grown cells, whereas three bands (Cat8pI, -II, and -III) were identified in derepressed cells. Derepression-specific Cat8pII and -III resulted from differential phosphorylation, as shown by phosphatase treatment. Only the most extensively phosphorylated modification (Cat8pIII) depended on the Cat1p (Snf1p) kinase, indicating that another protein kinase is responsible for modification form Cat8pII. The occurrence of Cat8pIII was strongly correlated with the derepression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) and gluconeogenic PCK1 mRNA. Furthermore, glucose triggered the dephosphorylation of Cat8pIII, but this did not depend on the Glc7p (Cid1p) phosphatase previously described as being involved in invertase repression. These results confirm our current model that glucose derepression of gluconeogenic genes needs Cat8p phosphorylation and additionally show that a still unknown transcriptional activator is also involved.

Cryopreservation of in vitro sugar beet shoot tips using the encapsulation-dehydration technique: influence of abscisic acid and cold acclimation.

It has been previously shown that shoot tips of in vitro plantlets of sugar beet (Beta vulgaris L. clone SES1) can be cryopreserved using the encapsulation-dehydration technique (survival rate of 37% after freezing). This article reports the influence of abscisic acid (ABA) and cold acclimation on survival after cryopreservation. When ABA was added to the multiplication medium of the plants, the survival rate of shoot tips after cryopreservation was not increased (45%). After cold acclimation of the plants, their growth pattern differed (plants became apically dominant) and the survival rate of the shoot tips after cryopreservation clearly increased (70% survival and 50% plant regeneration after freezing). This improved protocol was successfully applied to three other clones.

Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique.

Sugar beet shoot tips from cold-acclimated plants were successfully cryopreserved using a vitrification technique. Dissected shoot tips were precultured for 1 day at 5  °C on solidified DGJ0 medium with 0.3 M sucrose. After loading for 20 min with a mixture of 2 M glycerol and 0.4 M sucrose (20  °C), shoot tips were dehydrated with PVS2 (0  °C) for 20 min prior to immersion in liquid nitrogen. Both cold acclimation and loading enhanced the dehydration tolerance of shoot tips to PVS2. After thawing, shoot tips were deloaded for 15 min in liquid DGJ0 medium with 1.2 M sucrose (20  °C). The optimal exposure time to both loading solution and PVS2 depended on the in vitro morphology of the clone. With tetraploid clones a higher sucrose concentration during cold acclimation and preculture further enhanced survival after cryopreservation. Survival rates ranged between 60% and 100% depending on the clone. Since only 10-50% of the surviving shoot tips developed into non-hyperhydric shoots, regrowth was optimized.